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© 2019 by the authors; licensee Asian Online Journal Publishing Group 
 

Agriculture and Food Sciences Research 
Vol. 6, No. 1, 66-70, 2019 

ISSN(E) 2411-6653/ ISSN(P) 2518-0193 
DOI: 10.20448/journal.512.2019.61.66.70 

© 2019 by the authors; licensee Asian Online Journal Publishing Group 

    
 

 
 
Leaf Hexane Extracts of Two Turkish Fig (Ficus carica L.) Cultivars Show 
Cytotoxic Effects on a Human Prostate Cancer Cell Line 

 
Olcay Boyacioglu1    

Betül Kara2    

Hilal Can3      

Tugçe Naile Yerci4   

Sima Yilmaz5    

Seda Orenay Boyacioglu6    

 

 
( Corresponding Author) 

 
1,3,4,5Department of Food Engineering, Faculty of Engineering, Aydin Adnan Menderes University, Efeler, Aydin, 
09010, Turkey. 

 
2Department of Food Engineering, Faculty of Engineering, Aydin Adnan Menderes University, Efeler, Aydin, 
09010, Turkey; Molecular Biotechnology Program, Institute of Health Sciences, Aydin Adnan Menderes 
University, Efeler, Aydin, 09010, Turkey. 

 
6Department of Medical Genetics, Faculty of Medicine, Aydin Adnan Menderes University, Efeler, Aydin, 09010, 
Turkey. 

 

 
Abstract 

The world-famous Turkish fig, grown in Aydin, is also used for the treatment of various diseases 
with its leaves and latex. Studies have shown that fig leaf has antioxidant, antiviral, antidiabetic, 
antimicrobial, and anticancer effects. The objective of the study is to examine the anticarcinogenic 
and antimicrobial effects of nonpolar extracts from the leaves of two fig cultivars (Sari Lop and 
Aydin Black) that are the most widely grown in Aydin. The fig leaves collected in the summer of 
2017 were dried in shade at room temperature and crumbled. The n-hexane extracts of the dried 
fig leaves obtained with manual soxhlet and semi-automated soxhlet apparatus were applied on 
PC3 human prostate cancer cell line for 24h. The antimicrobial activities of the extracts were 
examined on Escherichia coli and Bacillus cereus using disc and agar well diffusion methods. As a 
result, antimicrobial activity of the n-hexane extracts on the bacteria was not detected at the 
highest dose tested (100 mg/ml). The n-hexane extracts showed cytotoxic effect on PC3 cells in a 

dose-dependent fashion and caused close to 100% death at 1000 μg/ml. No significant difference 
was observed between the cytotoxic effects of n-hexane extracts of two fig cultivars and the 
extraction methods (P>0.05). 

 
Keywords: Ficus carica, Fig leaf, Hexane extract, Prostate cancer, Cytotoxicity, Antimicrobial. 

 
Citation | Olcay Boyacioglu; Betül Kara; Hilal Can; Tugçe Naile 
Yerci; Sima Yilmaz; Seda Orenay Boyacioglu (2019) Leaf Hexane 
Extracts of Two Turkish Fig (Ficus carica L.) Cultivars Show 
Cytotoxic Effects on a Human Prostate Cancer Cell Line. 
Agriculture and Food Sciences Research, 6(1): 66-70. 
History:  
Received: 31 January 2019 
Revised: 7 March 2019 
Accepted: 17 April 2019 
Published: 11 June 2019 
Licensed: This work is licensed under a Creative Commons 

Attribution 3.0 License  
Publisher:  Asian Online Journal Publishing Group 

Contribution/Acknowledgement: All authors contributed to the conception 
and design of the study. Bacterial strains were generously gifted by Prof. Dr. 
H. Halil Bıyık, and the PC3 cell line by Prof. Dr. William H. Gmeiner. 
Funding: This work was supported by the Scientific and Technological 
Research Council of Turkey (TUBITAK), BIDEB 2209-A program, Grant 
number 1919B011700595 to BK.  
Competing Interests: The authors declare that they have no conflict of 
interests. 
Transparency: The authors confirm that the manuscript is an honest, 
accurate, and transparent account of the study was reported; that no vital 
features of the study have been omitted; and that any discrepancies from the 
study as planned have been explained. 
Ethical: This study follows all ethical practices during writing.   

 

 

Contents 
1. Introduction ...................................................................................................................................................................................... 67 
2. Materials and Methods ................................................................................................................................................................... 67 
3. Results and Discussion ................................................................................................................................................................... 68 
4. Conclusion ......................................................................................................................................................................................... 69 
References .............................................................................................................................................................................................. 69 
 

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1. Introduction 
Fig (Ficus carica L.), a fruit tree of the Moraceae family, is a small tree with a height ranging from 1 to 10 m. F. 

carica plant is grown mostly in the city of Aydin in Aegean Region of Turkey [1]. The fig is cultivated worldwide 
for its nutritive and medical properties. It is widely used in Ayurveda, siddha, and homeopathy medical systems 
[2]. Fruits, leaves, and roots of fig plants are some of the remedies people resort to in various illnesses. While fig 
fruit is used for the prevention of indigestion, anorexia, cardiovascular problems, and cancers; fig roots and leaves 
are used against indigestion and anorexia. In addition, fig leaf is also used against the anemia [3]. The total 
phenolic substance, organic acid content, and antioxidant potential in the fig leaf have been reported to be higher 
than in fig pulp and crusts [4].  

It is claimed that F. carica is useful in treating liver and spleen disorders and gout disease. Fig leaves were also 
tried for the treatment of jaundice. F. carica leaves boiled in hot water were reported to be used in kidneys and 
liver, in diabetes and calcification, and as well as in hemorrhoid treatment [5, 6]. F. carica leaves have also been 
used in the treatment of vitiligo due to the presence of furanocoumarins, psoralen, and daidzein [7]. The fig leaf is 
also reported to be used in asthma, cough, sexual disorders, diarrhea, hematuria, earache, toothache, migraine, eye 
problems, stomach problems, and scabies [8]. In traditional medicine, the use of F. carica leaf, fruit, and root is a 
reality due to the antibacterial, antifungal, antioxidant, and antiviral activities it has [3].  

In 1998, when the aqueous decoction of fig leaf was administered to Type I diabetic patients, hypoglycaemic 
effect was revealed as the postprandial (after the meal) glycemia decreased significantly in the patients [9]. 
Following this, Canal et al. showed that chloroform extract obtained from aqueous decoction of F. carica leaves was 
successfully used to decrease cholesterol levels and hyperglycemia on the rats with experimentally-induced  
diabetes [10].  

It was reported that fig leaf methanol extract (500 mg/kg) decreased the aspartate aminotransferase, alanine 
aminotransferase, total serum bilirubin, and malondialdehyde levels, and showed liver protective effect by reducing 
lipid peroxidation in the liver [11]. This activity of the fig leaf extract is comparable to slymarine [12] which is 
known as a liver protective agent.  

The petroleum ether extract of F. carica leaves provided promising results in the treatment of rifampicin-
induced hepatic damage in rats [13]. A study conducted on Wistar albino rats showed that the petroleum ether, 
chloroform, and ethanol extracts of F. carica leaves had anti-imflammatory properties [8]. In a recent study, ethyl 
acetate extract of fig leaf was shown to be controlling the glucose and fat levels in type 2 diabetic rats, thus being 
effective in controlling the carbohydrate metabolism [14]. While promising results were obtained with F. carica 
leaf extracts, ethanol extract of F. carica fruit (fig) up to 1000 ug/ml for 72 h did not show any toxicity on U87 
glioblastoma cells [15].  

In a study conducted in Iran, the anti-angiogenic effects of fig leaf ethanolic extract was evaluated on human 

umbilical vein endothelial cells (HUVEC). After 24 hours of incubation with the extract at 25 μg/ml, a statistically 
significant cytotoxic effect on HUVECs was reported compared to the control group [16].  

The antimicrobial effects of F. carica leaf extracts have been examined in several studies. The methanol extract 
of fig leaves collected and dried in South Korea was detected to have antimicrobial activity on oral bacteria [17]. In 
2016, total phenolic and flavonoid contents of the methanol extracts from ten different varieties of F. carica in 
Algeria were examined and antimicrobial effects of the extracts were determined by disk diffusion test. As a result, 
all extracts were rich in phenolic compounds and had antimicrobial effect against Gram-positive and Gram-
negative bacteria. Staphylococcus aureus and Bacillus cereus were reported to be the most susceptible bacteria to F. 
carica extracts [18]. In another study, the ethanol extract of fig leaf showed growth inhibitory effect against the 
bacteria Staphylococcus aureus, Salmonella typhi, and the mold Fusarium oxysporum. Also, fig latex was tried in this 
study and reported to have a stronger effect. Aspergillus niger and E. coli were more resistant to ethanolic extract 
and fig latex [19].  

To the best of our knowledge, no results were reported on the essential oil and n-hexane extracts of fig leaves. 
The objectives of this study, were to assess the antibacterial and antiproliferative effects of nonpolar extracts from 
leaf of two Turkish fig cultivars on two bacterial samples and on PC3 human prostate cancer cell line, respectively.  
 

2. Materials and Methods 
2.1. Preparation of Fig Leaves 

The research material fig leaves were collected from the F. carica cultivar Sari Lop trees grown in the Aydin 
Adnan Menderes University Central Campus (37°51'28.3"N 27°51'23.8"E) and F. carica cv. Aydin Black (also 
known as Bursa Black) leaves were collected from a fig orchard in Gencelli village of Kuyucak district in the city of 
Aydin (37°56'04.5"N 28°39'28.9"E) in the summer of 2017. The collected leaves were visually inspected not to have 
damage due to sunburn, plant pests, or human handling. The leaves were shade-dried at room temperature for a 
week and crushed. The resulting leaf pieces were stored in dry and cool room conditions.  
 
2.2. Determination of Water Content of the Fig Leaves 

Fresh, mature or young (leaf length < 10 cm) fig leaf pieces ranging between 4 and 10 gr was weighed into a 
pre-weighted crucible. After 3 hours at 105°C in lab oven, the crucible with the sample was cooled in a desiccator 
and weighed again to determine the water contents.  
 
2.3. Essential oil extraction  

Essential oil was extracted from 100 gr of crushed dry fig leaves mixed with 800 ml of distilled water by 6-hr 
hydrodistillation using the Clevenger-type apparatus.  
 
2.4. Hexane Extraction Methods 

In the first manual method, classical, glass soxhlet extraction system was used. Dried and crushed (24 gr) fig 
leaf samples in the cellulose thimbles were extracted with 300 ml of n-hexane for 2.5 hours. The resulting extract 



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was filtered twice through Whatman No. 1 filter paper and concentrated by rotary evaporator (Büchi Labortechnik 
AG, R-100, Flawil, Switzerland) at 55°C at about 70 bar vacuum pressure. The resulting solid extract mass was 
weighed, diluted in n-hexane to a concentration of 0.1 gr/ml (10%), and stored at -18°C.  

For the second extraction method, dried and crushed Sari Lop and Aydin Black fig leaves (a total of 24 gr in 3 
cellulose thimbles) were extracted with a total of 150 ml of n-hexane in a semi-automated soxhlet device (Velf 
Scienfitica, ser 148/3, 20865 Usmate, Italy). The extraction program is composed of 40 min immersion, 80 min 
washing, and 3 min recovery. At the end of the extraction, the leftover n-hexane was removed and the resulting 
solid extract mass was stored as described above.  
 
2.5. Bacterial Species 

Antimicrobial assays were performed on E. coli ATCC 35218 and B. cereus ATCC 11778 (gifted by Prof. Dr. H. 
Halil Bıyık, Aydın Adnan Menderes University). Bacteria were cultured in tryptic soy broth (TSB) (Merck) and 
tryptic soy agar (TSA) media consisting of TSB supplemented with 15% agar agar (Sigma Aldrich).  
 
2.6. Disc Diffusion and Agar Well Diffusion Methods 

Fifty μl of E. coli or B. cereus culture grown in TSB for 6-8 hours was transferred onto Mueller Hinton Agar 

(MHA) plates (Merck) and spread over the entire surface to form a thin film layer. Ten μl of the n-hexane extract 
of fig leaf dissolved in n-hexane or acetone at 0.1 gr/ml concentration was impregnated into sterile 6 mm 
antimicrobial susceptibility disks (Oxoid) on the surface of MHA plate, and the discs were pressed down gently to 

ensure complete contact with the agar surface. The n-hexane or acetone (10 μl) and a Gentamicin (Oxoid) disk 
were used as negative and positive controls, respectively. Plates were incubated at 37°C for up to 48 h and the 
bacterial growth inhibition zones around the discs were observed and measured.  

Agar well diffusion assay was used to increase the amount of extract tested. After the test bacteria were spread 
on the MHA plates as described in the disc diffusion method, wells with 5 mm in diameter were formed on the agar 
under aseptic conditions. Each well was filled with the n-hexane extract of fig leaf dissolved in n-hexane or acetone 
at 0.1 gr/ml concentration. Plates were incubated and the bacterial growth inhibition zones around the discs were 
observed as described above.  
 
2.7. Prostate Cancer Cell Line Culture 

The anti-carcinogenic properties of the n-hexane extracts of the fig leaves were tested on PC3 human prostate 
cancer cell line (gifted by Prof. Dr. William H. Gmeiner, Wake Forest University). Cells were cultured in RPMI-
1640 (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (Biowest) in the presence of 5% CO2 at 
37°C. Cells were routinely passaged using Trypsin (Sigma-Aldrich) when they reached 80-90% confluence.  
 
2.8. Cytotoxicity MTT Assay 

PC3 cells were seeded at a density of 3000 cells/well in 96-well plate and incubated in CO2 incubator. The 

following day, hexane extracts were applied to the cells at 250, 500, 750, and 1000 μg/ml concentrations for 24-48 
hours, and cell viabilities were measured colorimetrically by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium 
Bromide (MTT) (Sigma-Aldrich). MTT was dissolved in sterile phosphate buffered saline (PBS) and further 
diluted in RPMI with 10% FBS to a final MTT concentration of 0.5 mg/ml. At the end of the fig leaf extract 

treatment, the used media was replaced with 120 μl of new media with MTT. After a minimum 3 hours of 
incubation, the violet colored formazan crystals inside the cells were observed under a microscope, and the MTT 

containing media was replaced with 100 μl of dimethyl sulfoxide (DMSO) (Merck). After the plate was shaken for 
10 min to dissolve the formazan crystals, results were obtained with a spectrophotometer (Thermo Labsystems 
Multiskan Spectrum Mic) at 570 nm. All experiments were performed in triplicates and the experiments were 
repeated 3 times.  
 

3. Results and Discussion 
The water contents of fresh, mature fig leaves and the young leaves (leaf length < 10 cm) for both fig cultivars 

were found to be 70 and 78%, respectively. These values are correlated with the water content of mint leaves, 
which was reported as 70-80% [20].  

Essential oil extraction from both fig cultivar leaves was not possible as no essential oil was observed after the 
water distillation method using Clevenger apparatus, possibly due to low amount of essential oil in the leaves. To 
check the equipment functionality, essential oil of Thymus vulgaris L. was successfully extracted. A larger volumed 
extraction system might help in extracting essential oil from fig leaves.  

The n-hexane extraction yield rates of F. carica leaves in manual and semi-automated soxhlet methods were 
1.55 and 2.30 mg/gr, respectively for the Sari Lop cultivar and 2.15 and 2.40 mg/gr, respectively for the Aydin 
Black cultivar.  

First, the F. carica leaf extracts prepared at 10% concentration in n-hexane were used in disc diffusion tests 
Table 1. However, considering the low volume of extract used in discs and the fast vaporizing nature of n-hexane, 
no antibacterial effect of the extracts or the n-hexane negative control was observed. For this reason, the n-hexane 
extracts were solubilized in acetone at 10% concentration but no antimicrobial effect on the growth of E. coli and B. 
cereus was observed either Table 1. The negative control acetone showed greater antimicrobial effect than all of the 
extracts. None of the fig leaf extracts obtained by Semi-automated soxhlet system and dissolved in acetone had 
antimicrobial effect against B. cereus. Similar results were obtained in well diffusion tests (results not shown).  
 
 
 
 
 



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Table-1. Result of disc diffusion tests using the n-hexane extract of fig leaves dissolved in n-hexane or acetone. 

Extraction 
solvent 

Reagents tested Diameter of growth inhibition zone (mm) 

E. coli ATCC 35218 B. cereus ATCC 11778 

None Gentamicin 
(Pos. control) 

12 21 

n-hexane 

n-hexane 
(Neg. Control) 

6 6 

Sari Lop M 6 6 
Sari Lop S 6 6 

Aydin Black M 6 6 
Aydin Black S 6 6 

acetone 

Acetone 
(Neg. Control) 

15 15 

Sari Lop M 12 12 
Sari Lop S 8 6 

Aydin Black M 10 10 
Aydin Black S 13 6 

               Note: M: Manual soxhlet; S: Semi-automated soxhlet. 
 

However, a recent study conducted in Algeria reported that methanol extracts of 10 different varieties of F. 
carica leaves showed antimicrobial properties against Gram positive and Gram negative bacteria [18]. Type of 
extraction solvent used, geographic and climatic differences between Turkey and Algeria, and the type of the fig 
trees may be counted as the potential reasons for the difference in antimicrobial effectiveness between the studies. 
Another study conducted in South Korea with fig leaf methanol extract also reported antimicrobial effect against 
the oral bacteria [17]. In 1998, hypoglycemic effect of fig leaf was investigated in type I diabetic patients [9] and 
in 2000, a chloroform extract from F. carica leaves was used to reduce cholesterol levels in diabetic mice [21].  

In this study, cytotoxic effects of n-hexane extract of fig leaves collected from two local cultivars (Sari Lop and 
Aydin Black) grown in Aydin, Turkey were investigated on the PC3 human prostate cancer cell line. Fig leaf 
extracts were applied on cells at doses ranging from 0-1000 µg/ml for 24 h. Results reveal that fig leaf extracts 
showed cytotoxic effect on PC3 cells in a dose-dependent manner Figure 1. The leaf extracts started showing 50% 
cytotoxicity on PC3 cells after 24 h treatment at 250 µg/ml dose. Almost complete cytotoxicity was observed on 
PC3 cells after 24 h treatment at 1000 µg/ml. No statistically significant difference was available between the 
cytotoxic effects of Sari Lop and Aydin Black cultivars (t-test, P>0.05). Also, there was no significant difference in 
the comparison of the extraction type (manual soxhlet vs. semi-automated soxhlet device) (t-test, P>0.05).  
 

 
Figure-1. Treatment of n-hexane extracts of fig leaves on PC3 cells for 24 h. M: Manual soxhlet; S: Semi-automated soxhlet. 

 
The petroleum ether extract of F. carica leaf was successfully used in rats to protect the liver from damage by 

rifampicin [13]. Similarly, hepatoprotective effects of fig leaf ethanol [22] and methanol [23] extracts were 
reported against hepatotoxicity caused by carbon tetrachloride. Ethanol extract of fig leaf was reported to have a 
curing effect against both cellular and humoral antibody response in mice [24]. It was also indicated that fig leaf 
ethanol extract has immunity stimulating effect. Additionally, the water extract of fig leaf is known to have toxicity 
against herpes simplex virus [25].  
 

4. Conclusion 
In conclusion, F. carica fruit, latex, and leaf have been researched for their beneficial effects by various studies. 

This study revealed that 24 h treatment of n-hexane extract of two fig cultivars shows cytotoxic effect on PC3 
human prostate cancer cell line.  
 

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